Stage-specific expressions of four different ribonuclease H genes in Leishmania

The human pathogen of the genus Leishmania cause worldwide morbidity and infection of visceral organs by some species may be lethal. Lack of rational chemotherapy against these pathogens dictates the study of differential biochemistry and molecular biology of the parasite as compared to its human host. The ubiquitous enzyme ribonuclease H (RNase H, EC 3.1.26.4) cleaves the RNA from a RNA:DNA duplex and is critical for the replication of DNA in the nucleus and the mitochondria. We have characterized four RNase H genes from Leishmania: one is of type I (LRNase HI) and three others are of type II (LRNase HIIA, -HIIB and -HIIC). In contrast human cells have only one type I and one type II RNase H. All the four RNase H genes in Leishmania are single copy and located in discrete chromosomes. When expressed inside RNase H-deficient E. coli, all of the four Leishmania RNase H were capable to complement the genetic defect of the E. coli, indicating their identity as RNase H. The enzymes are differentially expressed in the promastigotes and the amastigotes, the forms that thrives in entirely different physico-chemical conditions in nature. Nucleotide sequences of the 5'-UTRs of three of these mRNAs have upstream open reading frames. Understanding the regulation of these four distinct ribonuclease H genes in Leishmania will help us better understand leishmanial parasitism and may help us to design rational chemotherapy against the pathogen

A Novel Vaccine Approach for Chagas Disease Using Rare Adenovirus Serotype 48 Vectors

Due to the increasing amount of people afflicted worldwide with Chagas disease and an increasing prevalence in the United States, there is a greater need to develop a safe and effective vaccine for this neglected disease. Adenovirus serotype 5 (Ad5) is the most common adenovirus vector used for gene therapy and vaccine approaches, but its efficacy is limited by preexisting vector immunity in humans resulting from natural infections. Therefore, we have employed rare serotype adenovirus 48 (Ad48) as an alternative choice for adenovirus/Chagas vaccine therapy. In this study, we modified Ad5 and Ad48 vectors to contain T. cruzi’s amastigote surface protein 2 (ASP-2) in the adenoviral early gene. We also modified Ad5 and Ad48 vectors to utilize the “Antigen Capsid-Incorporation” strategy by adding T. cruzi epitopes to protein IX (pIX). Mice that were immunized with the modified vectors were able to elicit T. cruzi-specific humoral and cellular responses. This study indicates that Ad48-modified vectors function comparable to or even premium to Ad5-modified vectors. This study provides novel data demonstrating that Ad48 can be used as a potential adenovirus vaccine vector against Chagas disease

Immunization with Hexon modified adenoviral vectors integrated with gp83 epitope provides protection against Trypanosoma cruzi infection.

Trypanosoma cruzi is the causative agent of Chagas disease. Chagas disease is an endemic infection that affects over 8 million people throughout Latin America and now has become a global challenge. The current pharmacological treatment of patients is unsuccessful in most cases, highly toxic, and no vaccines are available. The results of inadequate treatment could lead to heart failure resulting in death. Therefore, a vaccine that elicits neutralizing antibodies mediated by cell-mediated immune responses and protection against Chagas disease is necessary.The "antigen capsid-incorporation" strategy is based upon the display of the T. cruzi epitope as an integral component of the adenovirus' capsid rather than an encoded transgene. This strategy is predicted to induce a robust humoral immune response to the presented antigen, similar to the response provoked by native Ad capsid proteins. The antigen chosen was T. cruzi gp83, a ligand that is used by T. cruzi to attach to host cells to initiate infection. The gp83 epitope, recognized by the neutralizing MAb 4A4, along with His6 were incorporated into the Ad serotype 5 (Ad5) vector to generate the vector Ad5-HVR1-gp83-18 (Ad5-gp83). This vector was evaluated by molecular and immunological analyses. Vectors were injected to elicit immune responses against gp83 in mouse models. Our findings indicate that mice immunized with the vector Ad5-gp83 and challenged with a lethal dose of T. cruzi trypomastigotes confer strong immunoprotection with significant reduction in parasitemia levels, increased survival rate and induction of neutralizing antibodies.This data demonstrates that immunization with adenovirus containing capsid-incorporated T. cruzi antigen elicits a significant anti-gp83-specific response in two different mouse models, and protection against T. cruzi infection by eliciting neutralizing antibodies mediated by cell-mediated immune responses, as evidenced by the production of several Ig isotypes. Taken together, these novel results show that the recombinant Ad5 presenting T. cruzi gp83 antigen is a useful candidate for the development of a vaccine against Chagas disease

Antigen capsid-incorporation vector elicits an <i>in vivo T. cruzi</i> isotype-specific response.

<p>Post-prime, post-boost, and post-reboost of BALB/c and C57BL/6 mice (n = 7) serum was used for the isotype-specific assays. A–B) Ten µM of purified gp83 (KIYWKQPVEGTKSWTLSK) antigenic peptide was bound to ELISA plates. Residual unbound peptide was washed from the plates. The plates were then incubated with immunized mice serum followed by isotype-specific antibodies. The binding was detected with HRP conjugated secondary antibodies. OD at 450 nm represents isotype-specific <i>T. cruzi</i> gp83 antibody levels in sera. The values are expressed as the mean ± standard deviation. (*) = <i>P</i>≤0.05, and (**) = <i>P</i>≤0.01. C–D) Pie charts illustrating the isotype distribution patterns post-prime, post-boost, and post-reboost for immunized BALB/c mice or C57BL/6 sera.</p

Antigens are exposed on the virion surface.

<p>A) Varying amounts (starting at 4.5×10⁹ VP/mouse) of Ad5 or Ad5-gp83 were immobilized into the wells of ELISA plates and incubated with His₆ MAb. B) Either Ad5 or Ad5-gp83 at 6×10⁸ VP of were immobilized on an ELISA plate followed by varying dilutions of monoclonal antibody to His₆ (starting at 1∶2,000 dilution). The binding was detected with a HRP-conjugated secondary antibody. OD was read at 450 nm with a microplate reader.</p

Mice immunization with antigen capsid-incorporation vector protects against the challenge with <i>T. cruzi</i> and elicit neutralizing antibodies.

<p>A) Parasitemia of vaccinated mice. C57BL/6 mice (5 per group, 6-week old) that were immunized with either Ad5 or Ad5-p83 were challenged intraperitoneally with a lethal dose of blood trypomastigotes (5×10³) and the kinetics of parasitemia was determined in 5 µl of blood tail. Data represent the mean values ± SEM. B) Kaplan-Meier survival plot. C) Neutralization of <i>T. cruzi</i> infection of cardiomyocytes with Abs from vaccinated mice. Parasite multiplication within cell monolayers was estimated by determining the fluorescence level of parasites expressing green fluorescent protein, which is indicated as relative fluorescence units (RFU) at 72 hours of infection. Data represent the mean values ± SEM of the results from triplicate samples. (***) = <i>P</i>≤0.001. D) Fluorescence microscopic observation of the effect of neutralizing antibodies on cardiomyocyte infection by <i>T. cruzi</i>. Trypomastigotes expressing GFP were pre-treated with Abs and exposed to cardiomyocytes for 72 h as described in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003089#s2" target="_blank">Material and Methods</a>. Abs were obtained from vaccinated mice with either Ad or Ad5-gp83 before <i>T. cruzi</i> challenge. GFP-expressing amastigotes are seen inside host cells, host cell nuclei are stained blue, and cellular actin filaments are stained red.</p

<i>T. cruzi</i> gp83 epitope and His₆ epitope genetically incorporated into the HVR1 of Ad5.

<p>Rescued vectors were upscaled and viral DNA was isolated and analyzed to confirm the modification of the relevant genes. A) Schematic of adenoviral genomes. (1) Ad5, a replication-defective adenovirus with unmodified hexon. (2) Ad5-gp83, Ad5 replication-defective genome containing the hexon modification. B) Hexon-specific PCR primers confirmed the presence of the hexon gene in all of the vectors. Lane 1, DNA ladder, lane 2, Ad5; and lane 3, Ad5-gp83. C) <i>T. cruzi</i>-His₆-specific primers confirmed the incorporation of gp83 and His₆ epitopes. Lane 1, DNA ladder, lane 2, Ad5; and lane 3, Ad5-gp83. D) Western blot analysis confirmed the presence of His₆ incorporation within the modified vector. E) Western blot analysis confirmed the presence of fiber within the modified vector. In these assays, protein marker (lane 1), Ad5 (lane 2), and Ad5-gp83 (lane 3) were separated by 4–15% SDS-PAGE. The proteins were transferred to polyvinylidene fluoride (PVDF) membranes then incubated with either monoclonal antibodies to His₆ or fiber. The binding was detected with a HRP-conjugated secondary antibody.</p

Antigen capsid-incorporation vector elicits an <i>in vivo T. cruzi</i> humoral immune response.

<p>BALB/c and C57BL/6 mice (n = 7) were primed, boosted, and reboosted with 1×10¹⁰ VP of Ad vectors. A) Immunization timeline showing when immunizations were performed (solid arrows) and sera was collected (dashed arrows). B) Post-prime, post-boost, and post-reboost BALB/c serum or C) Post-prime, post-boost, and post-reboost C57BL/6 serum was collected for ELISA binding assays. Ten µM of purified gp83 (KIYWKQPVEGTKSWTLSK) antigenic peptide was bound to ELISA plates. The plates were then incubated with serial diluted concentrations of immunized mice serum and the binding antibodies were detected with HRP conjugated secondary antibody. The amount of anti-gp83 antibodies in the sera was calculated based on a standard curve of mouse IgG protein. The values are expressed as the mean ± standard deviation. (*) = <i>P</i>≤0.05, and (**) = <i>P</i>≤0.01.</p

Virological properties of vectors used in this study.

<p>Virological properties of vectors used in this study.</p